Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.

MagC is a NplC/P60-like member of the α-2-macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan.

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities.

Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

Bacterial secretins: Mechanisms of assembly and membrane targeting.

Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.

CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope.

The coordinated reformation of the nuclear envelope (NE) after mitosis re-establishes the structural integrity and the functionality of the nuclear compartment. The endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodeling pathway that is highly conserved in eukaryotes, has been recently involved in NE resealing by mediating the annular fusion of the nuclear membrane (NM). We show here that CC2D1B, a regulator of ESCRT polymerization, is required to re-establish the nuclear compartmentalization by coordinating endoplasmic reticulum (ER) membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE. Accordingly, CC2D1B determines the spatiotemporal distribution of the CHMP7-ESCRT-III axis during NE reformation. Crucially, in CC2D1B-depleted cells, ESCRT activity is uncoupled from Spastin-mediated severing of spindle microtubules, resulting in persisting microtubules that compromise nuclear morphology. Therefore, we reveal CC2D1B as an essential regulatory factor that licenses the formation of ESCRT-III polymers to ensure the orderly reformation of the NE.

Human viperin catalyzes the modification of GPP and FPP potentially affecting cholesterol synthesis.

Viperin is a radical SAM enzyme that possesses antiviral properties against a broad range of enveloped viruses. Here, we describe the activity of human viperin with two molecules of the mevalonate pathway, geranyl pyrophosphate, and farnesyl pyrophosphate, involved in cholesterol biosynthesis. We postulate that the radical modification of these two molecules by viperin might lead to defects in cholesterol synthesis, thereby affecting the composition of lipid rafts and subsequent enveloped virus budding.

Asymmetric ring structure of Vps4 required for ESCRT-III disassembly.

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Structural basis for ESCRT-III CHMP3 recruitment of AMSH.

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.

Streptococcal M1 protein constructs a pathological host fibrinogen network.

M1 protein, a major virulence factor of the leading invasive strain of group A Streptococcus, is sufficient to induce toxic-shock-like vascular leakage and tissue injury. These events are triggered by the formation of a complex between M1 and fibrinogen that, unlike M1 or fibrinogen alone, leads to neutrophil activation. Here we provide a structural explanation for the pathological properties of the complex formed between streptococcal M1 and human fibrinogen. A conformationally dynamic coiled-coil dimer of M1 was found to organize four fibrinogen molecules into a specific cross-like pattern. This pattern supported the construction of a supramolecular network that was required for neutrophil activation but was distinct from a fibrin clot. Disruption of this network into other supramolecular assemblies was not tolerated. These results have bearing on the pathophysiology of streptococcal toxic shock.

Divergent pathways lead to ESCRT-III-catalyzed membrane fission.

Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.

Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield.

Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.

Coiled-coil irregularities and instabilities in group A Streptococcus M1 are required for virulence.

Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the approximately 3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction.

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of beta-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a "trapped" covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-D-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the D-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of beta-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of beta-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.

Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins.

Beta-lactam antibiotics, including penicillins and cephalosporins, inhibit penicillin-binding proteins (PBPs), which are essential for bacterial cell wall biogenesis. Pathogenic bacteria have evolved efficient antibiotic resistance mechanisms that, in Gram-positive bacteria, include mutations to PBPs that enable them to avoid beta-lactam inhibition. Lactivicin (LTV; 1) contains separate cycloserine and gamma-lactone rings and is the only known natural PBP inhibitor that does not contain a beta-lactam. Here we show that LTV and a more potent analog, phenoxyacetyl-LTV (PLTV; 2), are active against clinically isolated, penicillin-resistant Streptococcus pneumoniae strains. Crystallographic analyses of S. pneumoniae PBP1b reveal that LTV and PLTV inhibition involves opening of both monocyclic cycloserine and gamma-lactone rings. In PBP1b complexes, the ring-derived atoms from LTV and PLTV show a notable structural convergence with those derived from a complexed cephalosporin (cefotaxime; 3). The structures imply that derivatives of LTV will be useful in the search for new antibiotics with activity against beta-lactam-resistant bacteria.

Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes.

Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful beta-lactam antibiotics. The increasing incidence of beta-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies.

Active site restructuring regulates ligand recognition in class A penicillin-binding proteins.

Bacterial cell division is a complex, multimolecular process that requires biosynthesis of new peptidoglycan by penicillin-binding proteins (PBPs) during cell wall elongation and septum formation steps. Streptococcus pneumoniae has three bifunctional (class A) PBPs that catalyze both polymerization of glycan chains (glycosyltransfer) and cross-linking of pentapeptidic bridges (transpeptidation) during the peptidoglycan biosynthetic process. In addition to playing important roles in cell division, PBPs are also the targets for beta-lactam antibiotics and thus play key roles in drug-resistance mechanisms. The crystal structure of a soluble form of pneumococcal PBP1b (PBP1b*) has been solved to 1.9 A, thus providing previously undescribed structural information regarding a class A PBP from any organism. PBP1b* is a three-domain molecule harboring a short peptide from the glycosyltransferase domain bound to an interdomain linker region, the transpeptidase domain, and a C-terminal region. The structure of PBP1b* complexed with beta-lactam antibiotics reveals that ligand recognition requires a conformational modification involving conserved elements within the cleft. The open and closed structures of PBP1b* suggest how class A PBPs may become activated as novel peptidoglycan synthesis becomes necessary during the cell division process. In addition, this structure provides an initial framework for the understanding of the role of class A PBPs in the development of antibiotic resistance.